Use of acid-stable subtilisin proteases in animal feed

ABSTRACT

Acid-stable proteases of the subtilisin family, their use in animal feed, feed-additives and feed compositions containing such proteases, and methods for the treatment of vegetable proteins using such proteases.

TECHNICAL FIELD

The present invention relates to the use of acid-stable, serineproteases of the subtilisin family in animal feed (in vivo), and to theuse of such proteases for treating vegetable proteins (in vitro).

Proteins are essential nutritional factors for animals and humans. Mostlivestock and many human beings get the necessary proteins fromvegetable protein sources. Important vegetable protein sources are e.g.oilseed crops, legumes and cereals.

When e.g. soybean meal is included in the feed of mono-gastric animalssuch as pigs and poultry, a significant proportion of the soybean mealsolids is not digested. E.g., the apparent ileal protein digestibilityin piglets and growing pigs is only around 80%.

The stomach of mono-gastric animals and many fish exhibits a stronglyacidic pH. Most of the protein digestion, however, occurs in the smallintestine. A need therefore exists for an acid-stable protease that cansurvive passage of the stomach.

BACKGROUND ART

The use of proteases in animal feed, or to treat vegetable proteins, isknown from the following documents:

WO95/28850 discloses i.a. an animal feed additive comprising a phytaseand a proteolytic enzyme. Various proteolytic enzymes are specified atp. 7.

WO96/05739 discloses an enzyme feed additive comprising xylanase and aprotease. Suitable proteases are listed at p. 25.

WO95/02044 discloses i.a. proteases derived from Aspergillus aculeatus,as well as the use in animal feed thereof.

U.S. Pat. No. 3,966,971 discloses a process of obtaining protein from avegetable protein source by treatment with an acid phytase andoptionally a proteolytic enzyme. Suitable proteases are specified incolumn 2.

U.S. Pat. Nos. 4,073,884, 5,047,240, 3,868,448, 3,823,072, and 3,683,069describe protease preparations derived from various strains ofStreptomyces and their use in animal feed.

These proteases, however, are not acid-stable and/or are not proteasesof the subtilisin family.

BRIEF DESCRIPTION OF THE INVENTION

Several proteases have now been identified which are found to be veryacid-stable, and expectedly of an improved performance in animal feed.These proteases belong to the group of proteases known as subtilisins.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to theaccompanying drawings, in which:

FIG. 1 shows pH-stability curves, viz. residual protease activity offour proteases (one acid-stable protease of the subtilisin familyderived from Bacillus sp. NCIMB 40484 (PD 498), and three referenceproteases (Sub.Novo, and Sub.Novo(Y217L), both derived from Bacillusamyloliquefaciens, and SAVINASE™) after incubation for 2 hours, at atemperature of 37° C., and at pH-values in the range of pH 2 to pH 11;the activity is relative to residual activity after a 2 hour incubationat pH 9.0, and 5° C.;

FIG. 2 shows pH-activity curves, viz. protease activity between pH 3 andpH 11, relative to the protease activity at pH-optimum, of the same fourproteases;

FIG. 3 shows temperature-activity curves at pH 9.0, viz. proteaseactivity at pH 9.0 between 15° C. and 80° C., relative to proteaseactivity at the optimum temperature, of the same four proteases;

FIG. 4 shows pH-stability curves similar to FIG. 1 but for five otheracid-stable proteases of the subtilisin family derived from Bacillusalcalophilus NCIMB 10438, Fusarium oxysporum IFO 4471, Paecilomyceslilacinus CBS 102449, Acremonium chrysogenum ATCC 48272, Acremoniumkiliense ATCC 20338;

FIG. 5 shows pH-activity curves similar to FIG. 2 but for the sameproteases as in FIG. 4; and

FIG. 6 shows temperature activity curves at pH 9.0 similar to FIG. 3 butfor the same proteases as in FIG. 4.

DETAILED DESCRIPTION OF THE INVENTION

The term protease as used herein is an enzyme that hydrolyses peptidebonds (has protease activity). Proteases are also called e.g.peptidases, proteinases, peptide hydrolases, or proteolytic enzymes.

Preferred proteases for use according to the invention are of theendo-type that act internally in polypeptide chains (endopeptidases).Endopeptidases show activity on N- and C-terminally blocked peptidesubstrates that are relevant for the specificity of the protease inquestion.

Included in the above definition of protease are any enzymes belongingto the EC 3.4 enzyme group (including each of the thirteensub-subclasses thereof) of the EC list (Enzyme Nomenclature 1992 fromNC-IUBMB, 1992), as regularly supplemented and updated.

Proteases are classified on the basis of their catalytic mechanism intothe following groupings: serine proteases (S), cysteine proteases (C),aspartic proteases (A), metalloproteases (M), and unknown, or as yetunclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J.Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), inparticular the general introduction part.

The term serine protease refers to serine peptidases and their clans asdefined in the above Handbook. In the 1998 version of this handbook,serine peptidases and their clans are dealt with in chapters 1-175.

In a particular embodiment, serine proteases are peptidases in which thecatalytic mechanism depends upon the hydroxyl group of a serine residueacting as the nucleophile that attacks the peptide bond.

The terms subtilisins or subtilisin family as used herein are intendedto include all Clan SB serine proteases, in particular Family S8 thereof(Clan SB is dealt with in Chapter 93 of the above handbook). Insubtilisins, the order of the catalytic triad is Asp-His-Ser. Thetertiary structure includes both alpha-helices and beta sheets. Clan SBincludes both endopeptidases and exopeptidases. These peptidases areknown from bacteria, archaea and eukaryotes; there is a singlerepresentative from a DNA virus.

For determining whether a given protease is a subtilisin or not,reference is made to the above Handbook and the principles indicatedtherein. Such determination can be carried out for all types ofproteases, be it naturally occurring or wild-type proteases; orgenetically engineered or synthetic proteases.

In the alternative, inhibition studies can be performed with SSI (theStreptomyces Subtilisin Inhibitor), and a subtilisin is defined as aprotease with up to 10% residual activity when inhibited with a molarexcess of SSI. This test may be carried out as described in Example 8.In particular embodiments of this definition, the subtilisin has up to8%, up to 6%, or up to 5% residual activity. The expression ‘up to’ isconsidered equal to the expression ‘less than or equal to’.

Protease activity can be measured using any assay, in which a substrateis employed, that includes peptide bonds relevant for the specificity ofthe protease in question. Assay-pH and assay-temperature are likewise tobe adapted to the protease in question. Examples of assay-pH-values arepH 5, 6, 7, 8, 9, 10, or 11. Examples of assay-temperatures are 25, 30,35, 37, 40, 45, 50, 55, 60, 65, or 70° C.

Examples of protease substrates are casein, and pNA-substrates, such asSuc-AAPF-pNA (available e.g. from Sigma S-7388). The capital letters inthis pNA-substrate refers to the one-letter amino acid code. Anotherexample is Protazyme AK (azurine-dyed crosslinked casein prepared astablets by Megazyme T-PRAK). For pH-activity and pH-stability studies,the pNA-substrate is preferred, whereas for temperature-activitystudies, the Protazyme AK substrate is preferred.

Examples of protease assays are described in the experimental part.

There are no limitations on the origin of the protease for use accordingto the invention. Thus, the term protease includes not only natural orwild-type proteases, but also any mutants, variants, fragments etc.thereof exhibiting protease activity, as well as synthetic proteases,such as shuffled proteases, and consensus proteases. Such geneticallyengineered proteases can be prepared as is generally known in the art,eg by Site-directed Mutagenesis, by PCR (using a PCR fragment containingthe desired mutation as one of the primers in the PCR reactions), or byRandom Mutagenesis. The preparation of consensus proteins is describedin eg EP 897985.

Examples of acid-stable proteases of the subtilisin family for useaccording to the invention are

(i) the proteases derived from Bacillus sp. NCIMB 40484, Bacillusalcalophilus NCIMB 10438; Fusarium oxysporum IFO 4471; Paecilomyceslilacinus CBS 102449, Acremonium chrysogenum ATCC 48272, and Acremoniumkiliense ATCC 20338;

(ii) proteases of at least 70, 75, 80, 85, 90, or at least 95% aminoacid identity to any of the proteases of (i);

(iii) proteases of at least 70, 75, 80, 85, 90, or at least 95% identityto any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4;

(iv) proteases of at least 70, 75, 80, 85, 90, or at least 95% aminoacid identity to any of SEQ ID NO: 5 (the whole sequence 1-397, orfragments 28-397 or 118-397 thereof), SEQ ID NO: 6 (the whole sequence1-367, or fragments 70-367 or 84-367 thereof), or SEQ ID NO: 7.

For calculating percentage identity, any computer program known in theart can be used, such as GAP provided in the GCG version 8 programpackage (Program Manual for the Wisconsin Package, Version 8, GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman,S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48,443-453. Using GAP with the following settings for polypeptide sequencecomparison: GAP creation penalty of 5.0 and GAP extension penalty of0.3.

In a particular embodiment, the protease for use according to theinvention is a microbial protease, the term microbial indicating thatthe protease is derived from, or originates from, a microorganism, or isan analogue, a fragment, a variant, a mutant, or a synthetic proteasederived from a microorganism. It may be produced or expressed in theoriginal wild-type microbial strain, in another microbial strain, or ina plant; i.e. the term covers the expression of wild-type, naturallyoccurring proteases, as well as expression in any host of recombinant,genetically engineered or synthetic proteases.

The term microorganism as used herein includes Archaea, bacteria, fungi,vira etc.

Examples of microorganisms are bacteria, such as bacteria of the genusBacillus, e.g. Bacillus sp. NCIMB No. 40484; Bacillus alcalophilus NCIMB10438; or mutants or variants thereof exhibiting protease activity.

Further examples of microorganisms are fungi, such as yeast orfilamentous fungi, e.g. chosen from the genera Paecilomyces, e.g.Paecilomyces lilacinus CBS 102449, Aspergillus, e.g. Aspergillus sp. CBS102448, Acremonium, e.g. Acremonium chrysogenum ATCC 48272, Acremoniumkiliense ATCC 20338, or Fusarium, e.g. Fusarium oxysporum IFO 4471; ormutants or variants thereof exhibiting protease activity.

In another embodiment the protease is a plant protease. An example of aprotease of plant origin is the protease from the sarcocarp of melonfruit (Kaneda et al, J. Biochem. 78, 1287-1296 (1975).

The term animal includes all animals, including human beings. Examplesof animals are non-ruminants, and ruminants, such as cows, sheep andhorses. In a particular embodiment, the animal is a non-ruminant animal.Non-ruminant animals include mono-gastric animals, e.g. pigs or swine(including, but not limited to, piglets, growing pigs, and sows);poultry such as turkeys and chicken (including but not limited tobroiler chicks, layers); young calves; and fish (including but notlimited to salmon).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the protease can be fed to theanimal before, after, or simultaneously with the diet. The latter ispreferred.

In the present context, the term acid-stable means, that the proteaseactivity of the pure protease enzyme, in a dilution corresponding toA₂₈₀=1.0, and following incubation for 2 hours at 37° C. in thefollowing buffer:

-   -   100 mM succinic acid, 100 mM HEPES, 100 mM CHES,    -   100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton® X-100, pH        3.5,        is at least 40% of the reference activity, as measured using the        assay described in Example 2C herein (substrate: Suc-AAPF-pNA,        pH 9.0, 25° C.)

In particular embodiments of the above acid-stability definition, theprotease activity is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, or atleast 90% of the reference activity.

The term reference activity refers to the protease activity of the sameprotease, following incubation in pure form, in a dilution correspondingto A₂₈₀=1.0, for 2 hours at 5° C. in the following buffer: 100 mMsuccinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl₂, 150mM KCl, 0.01% Triton® X-100, pH 9.0, wherein the activity is determinedas described above.

In other words, the method of determining acid-stability comprises thefollowing steps:

a) The protease sample to be tested (in pure form, A₂₈₀=1.0) is dividedin two aliquots (I and II);

b) Aliquot I is incubated for 2 hours at 37° C. and pH 3.5;

c) Residual activity of aliquot I is measured (pH 9.0 and 25° C.);

d) Aliquot II is incubated for 2 hours at 5° C. and pH 9.0;

e) Residual activity of aliquot II is measured (pH 9.0 and 25° C.);

f) Percentage residual activity of aliquot I relative to residualactivity of aliquot II is calculated.

Alternatively, in the above definition of acid-stability, the step b)buffer pH-value may be 2.0, 2.5, 3.0, 3.1, 3.2, 3.3, or 3.4.

In other alternative embodiments of the above acid-stability definitionrelating to the above alternative step b) buffer pH-values, the residualprotease activity as compared to the reference, is at least 5, 10, 15,20, 25, 30, 35, 40, 45, or at least 50%.

In alternative embodiments, pH values of 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5can be applied for the step d) buffer.

In the above acid-stability definition, the term A₂₈₀=1.0 means suchconcentration (dilution) of said pure protease which gives rise to anabsorption of 1.0 at 280 nm in a 1 cm path length cuvette relative to abuffer blank.

And in the above acid-stability definition, the term pure proteaserefers to a sample with a A₂₈₀/A₂₆₀ ratio above or equal to 1.70 (seeExample 2E), and which by a scan of a Coomassie-stained SDS-PAGE gel ismeasured to have at least 95% of its scan intensity in the bandcorresponding to said protease (see Example 2A). In the alternative, theA₂₈₀/A₂₆₀ ratio is above or equal to 1.50, 1.60, 1.65, 1.70, 1.75, 1.80,1.85, or above or equal to 1.90.

However, for the uses according to the invention, the protease need notbe that pure; it may e.g. include other enzymes, even other proteases,in which case it could be termed a protease preparation. Nevertheless, awell-defined protease preparation is advantageous. For instance, it ismuch easier to dose correctly to the feed a protease that is essentiallyfree from interfering or contaminating other proteases. The term dosecorrectly refers in particular to the objective of obtaining consistentand constant results, and the capability of optimising dosage based uponthe desired effect.

In a particular embodiment, the protease, in the form in which it isadded to the feed, or when being included in a feed additive, iswell-defined. Well-defined means that the protease preparation is atleast 50% pure as determined by Size-exclusion chromatography (seeExample 12).

In other particular embodiments the protease preparation is at least 60,70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by thismethod.

In the alternative, the term well-defined means, that a fractionation ofthe protease preparation on an appropriate Size-exclusion column revealsonly one major protease component.

The skilled worker will know how to select an appropriate Size-exclusionchromatography column. He might start by fractionating the preparationon e.g. a HiLoad26/60 Superdex75pg column from Amersham PharmaciaBiotech (see Example 12). If the peaks would not be clearly separated hewould try different columns (e.g. with an amended column particle sizeand/or column length), and/or he would amend the sample volume. Bysimple and common trial-and-error methods he would thereby arrive at acolumn with a sufficient resolution (clear separation of peaks), on thebasis of which the purity calculation is performed as described inExample 12.

The protease preparation can be (a) added directly to the feed (or useddirectly in the treatment process of vegetable proteins), or (b) it canbe used in the production of one or more intermediate compositions suchas feed additives or premixes that is subsequently added to the feed (orused in a treatment process). The degree of purity described aboverefers to the purity of the original protease preparation, whether usedaccording to (a) or (b) above.

Protease preparations with purities of this order of magnitude are inparticular obtainable using recombinant methods of production, whereasthey are not so easily obtained and also subject to a much higherbatch-to-batch variation when the protease is produced by traditionalfermentation methods.

Such protease preparation may of course be mixed with other enzymes.

In one particular embodiment, the protease for use according to theinvention, besides being acid-stable, also has a pH-activity optimumclose to neutral.

The term pH-activity optimum close to neutral means one or more of thefollowing: That the pH-optimum is in the interval of pH 6.0-11.0, or pH7.0-11.0, or pH 6.0-10.0, or pH 7.0-10.0, or pH 8.0-11.0, or pH 8.0-10.0(see Examples 2B and 7, and FIGS. 2 and 5 herein).

In another particular embodiment, the protease for use according to theinvention, besides being acid-stable, is also thermostable.

The term thermostable means one or more of the following: That thetemperature optimum is at least 50° C., 52° C., 54° C., 56° C., 58° C.,60° C., 62° C., 64° C., 66° C., 68° C., or at least 70° C., referencebeing made to Examples 2D and 7 and FIGS. 3 and 6 herein.

In a further particular embodiment, the protease for use according tothe invention is capable of solubilising vegetable proteins according tothe in vitro model of Example 4 herein.

The term vegetable proteins as used herein refers to any compound,composition, preparation or mixture that includes at least one proteinderived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g. soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, and sorghum.

The treatment according to the invention of vegetable proteins with atleast one acid-stable protease of the subtilisin family results in anincreased solubilisation of vegetable proteins.

The following are examples of % solubilised protein obtainable using theproteases of the invention: At least 76.8%, 77.0%, 77.2%, 77.4%, 77.6%,77.8%, 78.0%, 78.2%, 78.4%, 20 78.6%, or at least 78.8%, reference beinghad to the in vitro model of Example 4 herein.

The term solubilisation of proteins basically means bringing protein(s)into solution. Such solubilisation may be due to protease-mediatedrelease of protein from other components of the usually complex naturalcompositions such as feed. Solubilisation can be measured as an increasein the amount of soluble proteins, by reference to a sample with noprotease treatment (see Example 4 herein).

In a particular embodiment of a treatment process the protease(s) inquestion is affecting (or acting on, or exerting its solubilisinginfluence on the vegetable proteins or protein sources. To achieve this,the vegetable protein or protein source is typically suspended in asolvent, eg an aqueous solvent such as water, and the pH and temperaturevalues are adjusted paying due regard to the characteristics of theenzyme in question. For example, the treatment may take place at apH-value at which the relative activity of the actual protease is atleast 50, or 60, or 70, or 80 or 90%. Likewise, for example, thetreatment may take place at a temperature at which the relative activityof the actual protease is at least 50, or 60, or 70, or 80 or 90% (theserelative activities being defined as in Example 2 herein). The enzymaticreaction is continued until the desired result is achieved, followingwhich it may or may not be stopped by inactivating the enzyme, e.g. by aheat-treatment step.

In another particular embodiment of a treatment process of theinvention, the protease action is sustained, meaning e.g. that theprotease is added to the vegetable proteins or protein sources, but itssolubilising influence is so to speak not switched on until later whendesired, once suitable solubilising conditions are established, or onceany enzyme inhibitors are inactivated, or whatever other means couldhave been applied to postpone the action of the enzyme.

In one embodiment the treatment is a pre-treatment of animal feed orvegetable proteins for use in animal feed, i.e. the proteins aresolubilised before intake.

The term improving the nutritional value of an animal feed meansimproving the availability of the proteins, thereby leading to increasedprotein extraction, higher protein yields, and/or improved proteinutilisation. The nutritional value of the feed is therefore increased,and the growth rate and/or weight gain and/or feed conversion (i.e. theweight of ingested feed relative to weight gain) of the animal is/areimproved.

In particular embodiments the weight gain is at least 101%, 102%, 103%,104%, 105%, 106%, or at least 106.6% of the control, reference being hadto Example 10 herein.

In further particular embodiments the feed conversion is at most (or notmore than) 99%, 98%, 97.5%, 97%, or at most 96.6%. This is equivalent toa feed conversion of up to 99%, 98%, 97.5%, 97%, or up to 96.6%. Again,reference is had to Example 10 herein, comparing with the control.

The protease can be added to the feed in any form, be it as a relativelypure protease, or in admixture with other components intended foraddition to animal feed, i.e. in the form of animal feed additives, suchas the so-called pre-mixes for animal feed.

Animal Feed Additives

Apart from the acid-stable protease of the subtilisin family, the animalfeed additives of the invention contain at least one fat-solublevitamin, and/or at least one water-soluble vitamin, and/or at least onetrace mineral, and/or at least one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, aromacompounds, stabilisers, and/or at least one other enzyme selected fromamongst phytases EC 3.1.3.8 or 3.1.3.26; xylanases EC 3.2.1.8;galactanases EC 3.2.1.89; and/or beta-glucanases EC 3.2.1.4 (EC refersto Enzyme Classes according to Enzyme Nomenclature 1992 from NC-IUBMB,1992).

In a particular embodiment these other enzymes are well-defined (asdefined and exemplified above for protease preparations, i.a. byreference to Example 12).

Usually fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. Either of thesecomposition types, when enriched with an acid-stable subtilisinaccording to the invention, is an animal feed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01-10.0%; more particularly0.05-5.0%; or 0.2-1.0% (% meaning g additive per 100 g feed). This is soin particular for premixes.

Accordingly, the concentrations of the individual components of theanimal feed additive, e.g. the premix, can be found by multiplying thefinal in-feed concentration of the same component by, respectively,10-10000; 20-2000; or 100-500 (referring to the above three percentageinclusion intervals).

Guidelines for desired final concentrations, i.e.in-feed-concentrations, of such individual feed and feed additivecomponents are indicated in Table A below.

The following are non-exclusive lists of examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g. vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g. Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components—exemplified withpoultry and piglets/pigs—are listed in Table A below. Nutritionalrequirement means that these components should be provided in the dietin the concentrations indicated. These data are compiled from:

NRC, Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C. 1988; and

NRC, Nutrient requirements of poultry, ninth revised edition 1994,subcommittee on poultry nutrition, committee on animal nutrition, boardof agriculture, national research council. National Academy Press,Washington, D.C. 1994.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A. At leastone means either of, one or more of, one, or two, or three, or four andso forth up to all thirteen, or up to all fifteen individual components.

More specifically, this at least one individual component is included inthe additive of the invention in such an amount as to provide anin-feed-concentration within the range indicated in column four, orcolumn five, or column six of Table A.

As explained above, corresponding feed additive concentrations can befound by multiplying the interval limits of these ranges with 10-10000;20-2000; or 100-500. As an example, considering which premix-content ofvitamin A would correspond to the feed-content of 10-10000 IU/kg, thisexercise would lead to the following intervals: 100-10⁸ IU; or 200-2×10⁷IU; or 1000-5×10⁶ IU per kg additive.

TABLE A Nutrient requirements - and preferred ranges Nutrients providedper Piglets/ kg diet Poultry Pigs/Sows Range 1 Range 2 Range 3Fat-soluble vitamins Vitamin −5000 1300-4000   10-10000  50-8000 100-6000 A/[IU] Vitamin −1100 150-200   2-3000   5-2000  10-1500D₃/[IU] Vitamin −12 11-22 0.02-100  0.2-80  0.5-50  E/[IU] Vitamin0.5-1.5 −0.5 0.005-10.0  0.05-5.0  0.1-3.0 K/[mg] Water- solublevitamins B₁₂/[mg] −0.003 0.005-0.02  0.0001-1.000  0.0005-0.500 0.001-0.100 Biotin/[mg] 0.100-0.25  0.05-0.08 0.001-10.00 0.005-5.00 0.01-1.00 Choline/[mg]  800-1600 300-600   1-10000   5-5000  10-3000Trace minerals Manga- −60 2.0-4.0   0.1-1000  0.5-500  1.0-100 nese/[mg]Zinc/[mg] 40-70  50-100   1-1000  5-500  10-300 Iron/[mg] 50-80  40-100  1-1000  5-500  10-300 Copper/[mg] 6-8 3.0-6.0   0.1-1000  0.5-1001.0-25  Iodine/[mg] −0.4 −0.14 0.01-100  0.05-10   0.1-1.0 Sele- −0.20.10-0.30 0.005-100   0.01-10.0 0.05-1.0  nium/[mg] Macro mineralsCalcium/[g]  8-40 5-9 0.1-200  0.5-150  1-100 Phosphorus, 3-6 1.5-6  0.1-200  0.5-150  1-50 as available phospho- rus/[g]Animal Feed Compositions

Animal feed compositions or diets have a relatively high content ofprotein. According to the National Research Council (NRC) publicationsreferred to above, poultry and pig diets can be characterised asindicated in Table B below, columns 2-3. Fish diets can be characterisedas indicated in column 4 of Table B. Furthermore such fish diets usuallyhave a crude fat content of 200-310 g/kg. These fish diet areexemplified with diets for Salmonids and designed on the basis ofAquaculture, principles and practices, ed. T. V. R. Pillay, BlackwellScientific Publications Ltd. 1990; Fish nutrition, second edition, ed.John E. Halver, Academic Press Inc. 1989.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least oneprotease as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B below(R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e. Crude protein (g/kg)=N (g/kg)×6.25 as stated in Animal Nutrition,4th edition, Chapter 13 (Eds. P. McDonald, R. A. Edwards and J. F. D.Greenhalgh, Longman Scientific and Technical, 1988, ISBN 0-582-40903-9).The nitrogen content is determined by the Kjeldahl method (A.O.A.C.,1984, Official Methods of Analysis 14th ed., Association of OfficialAnalytical Chemists, Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient Requirements of Swine (1988) pp. 2-6, and theEuropean Table of Energy Values for Poultry Feed-stuffs, Spelderholtcentre for poultry research and extension, 7361 DA Beekbergen, TheNetherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one vegetable protein or protein source as definedabove.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-10% fish meal; and/or 0-20% whey.

Animal diets can e.g. be manufactured as mash feed (non-pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question. Enzymes canbe added as solid or liquid enzyme formulations. For example, a solidenzyme formulation is typically added before or during the mixing step;and a liquid enzyme preparation is typically added after the pelletingstep. The enzyme may also be incorporated in a feed additive or premix.The final enzyme concentration in the diet is within the range of0.01-200 mg enzyme protein per kg diet, for example in the range of 5-30mg enzyme protein per kg animal diet.

Examples of animal feed compositions are shown in Example 11.

TABLE B Range values for energy, protein and minerals in animal dietsPoul- try Piglets/ Fish Min- Pigs/Sows Min- Nutrient Max Min-Max Max R.1 R. 2 R. 3 R. 4 R. 5 Metabo- 12.1-13.4 12.9-13.5 14-25 10-30 11-2811-26 12-25 lisable energy, MJ/kg Crude 124-280 120-240 300-480  50-800 75-700 100-600 110-500 120-490 protein, g/kg Calcium,  8-40 5-9 10-15 0.1-200  0.5-150  1-100  4-50 g/kg Avail- 2.1-6.0 1.5-5.5  3-12 0.1-200  0.5-150  1-100  1-50  1-25 able Phospho- rus, g/kg Methio-3.2-5.5 — 12-16  0.1-100 0.5-75   1-50  1-30 nine, g/kg Methio- 4-92.3-6.8 —  0.1-150  0.5-125  1-80 nine plus Cys- teine, g/kg Lysine,2.5-11   6-14 12-22 0.5-50  0.5-40   1-30 g/kg

In particular embodiments of the method of the invention for treatingvegetable proteins, a further step of adding phytase is also included.And in further particular embodiments, in addition to the combinedtreatment with phytase and protease, further enzymes may also be added,wherein these enzymes are selected from the group comprising otherproteases, phytases, lipolytic enzymes, and glucosidase/carbohydraseenzymes. Examples of such enzymes are indicated in WO95/28850.

The protease should of course be applied in an effective amount, i.e. inan amount adequate for improving solubilisation and/or improvingnutritional value of feed. It is at present contemplated that the enzymeis administered in one or more of the following amounts (dosage ranges):0.01-200; or 0.01-100; or 0.05-100; or 0.05-50; or 0.10-10-all theseranges being in mg protease protein per kg feed (ppm).

For determining mg protease protein per kg feed, the protease ispurified from the feed composition, and the specific activity of thepurified protease is determined using a relevant assay (see underprotease activity, substrates, and assays). The protease activity of thefeed composition as such is also determined using the same assay, and onthe basis of these two determinations, the dosage in mg protease proteinper kg feed is calculated.

The same principles apply for determining mg protease protein in feedadditives.

Of course, if a sample is available of the protease used for preparingthe feed additive or the feed, the specific activity is determined fromthis sample (no need to purify the protease from the feed composition orthe additive).

Many vegetables contain anti-nutritional factors such as lectins andtrypsin inhibitors. The most important anti-nutritional factors ofsoybean are the lectin soybean agglutinin (SBA), and the soybean trypsininhibitor (STI).

Lectins are proteins that bind to specific carbohydrate-containingmolecules with considerable specificity, and when ingested they becomebound to the intestinal epithelium. This may lead to reduced viabilityof the epithelial cells and reduced absorption of nutrients.

SBA is a glycosylated, tetrameric lectin with a subunit molecular weightof about 30 kDa and a high affinity for N-acetylgalactosamine.

Trypsin inhibitors affect the intestinal proteolysis reducing proteindigestibility, and also increase the secretion of digestive enzymes fromthe pancreas leading to a loss of amino acids in the form of digestiveenzymes. An example of a trypsin inhibitor is the Bowman-Birk Inhibitor,that has a molecular weight of about 8 kDa, contains 7 disulfide bridgesand has two inhibitory loops specific for trypsin-like andchymotrypsin-like proteases. Other examples are the so-called KunitzInhibitors of Factors (e.g. the Soybean Kunitz Trypsin Inhibitor thatcontains one binding site for trypsin-like proteases and has a molecularweight of about 20 kDa).

The proteases for use according to the invention have been shown tohydrolyse anti-nutritional factors like SBA lectin, and the trypsininhibitors Bowman Birk Inhibitor and The Soybean Kunitz Factor. See theexperimental part, Example 5.

Thus, the invention also relates to the use of acid-stable serineproteases for hydrolysing, or reducing the amount of, anti-nutritionalfactors, e.g. SBA lectin, and trypsin inhibitors, such as the BowmanBirk Inhibitor, and Kunitz Factors, such as the Soybean Kunitz Factor.

EXAMPLE 1 Screening for Acid-stable Proteases

A large number of proteases were analysed for stability at pH 3, withthe objective of identifying proteases that have the necessary stabilityto pass through the acidic stomach of mono-gastric animals.

The proteases had been purified by conventional chromatographic methodssuch as ion-exchange chromatography, hydrophobic interactionchromatography and size exclusion chromatography (see e.g. ProteinPurification, Principles, High Resolution Methods, and Applications.Editors: Jan-Christer Janson, Lars Rydén, VCH Publishers, 1989).

Protease activity was determined as follows: The protease was incubatedwith 1.67% Hammarsten casein at 25° C., pH 9.5 for 30 minutes, then TCA(tri-chloro acetic acid) was added to a final concentration of 2% (w/w),the mixture was filtrated to remove the sediment, and the filtrate wasanalysed for free primary amino groups (determined in a colometric assaybased on OPA (o-phthal-dialdehyde) by measuring the absorbance at 340nm, using a serine standard (Biochemische Taschenbuch teil II,Springer-Verlag (1964), p.93 and p.102). One Casein Protease Unit (CPU)is defined as the amount of enzyme liberating 1 mmol of TCA-solubleprimary amino groups per minute under standard conditions, i.e. 25° C.and pH 9.5.

The proteases were diluted to an activity of 0.6 CPU/l in water, dividedin two aliquots and each aliquot was then further diluted to 0.3 CPU/lwith 100 mM citrate buffer, pH 3, and 100 mM phosphate buffer, pH 7respectively. The diluted samples were incubated at 37° C. for 1 hour,and 20 μl of the samples were applied to holes in 1% agarose platescontaining 1% skim milk. The plates (pH 7.0) were incubated at 37° C.over night and clearing zones were measured.

42 proteases performed well in this test. A number of these have beencharacterised, see examples 2, 6, 7 and 8. These proteases all belong tothe subtilisin family of serine proteases.

EXAMPLE 2 Characterisation and Comparative Study of the SubtilisinProtease Derived from Bacillus sp. NCIMB 40484

The protease derived from Bacillus sp. NCIMB 40484 was prepared asdescribed in Example 1 of WO93/24623.

The purpose of this characterisation was to study its pH-stability,pH-activity and temperature-activity profiles, in comparison toSub.Novo, Sub.Novo(Y217L), and SAVINASE™.

Sub.Novo is subtilisin from Bacillus amyloliquefaciens, andSub.Novo(Y217L) is the mutant thereof that is disclosed in WO96/05739.Sub.Novo was prepared and purified from a culture of the wild-typestrain using conventional methods, whereas the mutant was prepared asdescribed in Examples 1-2, and 15-16 of EP 130756.

SAVINASE™ is a subtilisin derived from Bacillus clausii (previouslyBacillus lentus NCIB 10309), commercially available from Novozymes A/S,Krogshoejvej, DK-2880 Bagsvaerd, Denmark. Its preparation is describedin U.S. Pat. No. 3,723,250.

EXAMPLE 2A Determination of SDS-PAGE Purity of Protease Samples

The SDS-PAGE purity of the protease samples was determined by thefollowing procedure:

40 μl protease solution (A₂₈₀ concentration=0.025) was mixed with 10 μl50% (w/v) TCA (trichloroacetic acid) in an Ep-pendorf tube on ice. Afterhalf an hour on ice the tube was centrifuged (5 minutes, 0° C.,14.000×g) and the supernatant was carefully removed. 20 μl SDS-PAGEsample buffer (200 μl Tris-Glycine SDS Sample Buffer (2×) (125 mMTris/HCl, pH 6.8, 4% (w/v) SDS, 50 ppm bromophenol blue, 20% (v/v)Glycerol, LC2676 from NOVEX™)+160 μl dist. water+20 μlβ-mercaptoethanol+20 μl 3M unbuffered Tris Base (Sigma T-1503) was addedto the precipitate and the tube was boiled for 3 minutes. The tube wascentrifuged shortly and 10 μl sample was applied to a 4-20% gradientTris-Glycine precast gel from NOVEX™ (polyacrylamide gradient gel basedon the Laemmli chemistry but without SDS in the gel, (Laemmli, U. K.,(1970) Nature, vol. 227, pp. 680-685), EC60255). The electrophoresis wasperformed with Tris-Glycine running buffer (2.9 g Tris Base, 14.4 gGlycine, 1.0 g SDS, distilled water to 1 liter) in both bufferreservoirs at a 150V constant voltage until the bromophenol bluetracking dye had reached the bottom of the gel. After electrophoresis,the gel was rinsed 3 times, 5 minutes each, with 100 ml of distilledwater by gentle shaking. The gel was then gently shaked with GelcodesBlue Stain Reagent (colloidal Comassie G-250 product from PIERCE, PIERCEcat. No. 24592) for one hour and washed by gentle shaking for 8 to 16hours with distilled water with several changes of distilled water.Finally, the gel was dried between 2 pieces of cellophane. Dried gelswere scanned with a Arcus II scanner from AGFA equipped with Fotolook 95v2.08 software and imported to the image evaluation software CREAM™ forWindows (catalogue nos. 990001 and 990005, Kem-En-Tec, Denmark) by theFile/Acquire command with the following settings (of Fotolook 95 v2.08):Original=Reflective, Mode=Color RGB, Scan resolution=240 ppi, Outputresolution=120 lpi, Scale factor=100%, Range=Histogram with Globalselection and Min=0 and Max=215, ToneCurve=None, Sharpness=None,Descreen=None and Flavor=None, thereby producing an *.img picture fileof the SDS-PAGE gel, which was used for evaluation in CREAM™. The *.imgpicture file was evaluated with the menu command Analysis/1-D. Two scanlines were placed on the *.img picture file with the Lane Place Tool: ASample scan line and a Background scan line. The Sample scan line wasplaced in the middle of a sample lane (with the protease in question)from just below the application slot to just above the position of theBromphenol blue tracking dye. The Background scan line was placedparallel to the Sample scan line, but at a position in the picturedSDS-PAGE gel where no sample was applied, start and endpoints for theBackground scan line were perpendicular to the start and endpoints ofthe Sample scan line. The Background scan line represents the truebackground of the gel. The width and shape of the scan lines were notadjusted. The intensity along the scan lines where now recorded with the1-D/Scan menu command with Medium sensitivity. Using the 1-D/Editor menucommand, the Background scan was subtracted from the Sample scan. Thenthe 1-D/Results menu command was selected and the Area % of the proteasepeak, as calculated by the CREAM™ software, was used as the SDS-PAGEpurity of the proteases.

The following results were obtained:

SDS-PAGE Protease Purity (Area %) From Bacillus sp. NCIMB 40484 96.3Sub. Novo 95.5 Sub. Novo (Y217L) 96.0 Savinase ® 99.2

EXAMPLE 2B pH-activity Assay

Suc-AAPF-pNA (Sigma® S-7388) was used for obtaining pH-activityprofiles.

Assay buffer: 100 mM succinic acid (Merck 1.00682), 100 mM HEPES (SigmaH-3375), 100 mM CHES (Sigma C-2885), 100 mM CABS (Sigma C-5580), 1 mMCaCl₂, 150 mM KCl, 0.01% Triton® X-100, adjusted to pH-values 3.0, 4.0,5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or 11.0 with HCl or NaOH.

Assay temperature: 25° C.

A 300 μl protease sample (diluted in 0.01% Triton® X-100) was mixed with1.5 ml of the assay buffer at the respective pH value, bringing the pHof the mixture to the pH of the assay buffer. The reaction was startedby adding 1.5 ml pNA substrate (50mg dissolved in 1.0 ml DMSO andfurther diluted 45× with 0.01% Triton X-100) and, after mixing, theincrease in A₄₀₅ was monitored by a spectrophotometer as a measurementof the protease activity at the pH in question. The assay was repeatedwith the assay buffer at the other pH values, and the activitymeasurements were plotted as relative activity against pH. The relativeactivities were normalized with the highest activity (pH-optimum), i.e.setting activity at pH-optimum to 1, or to 100%. The protease sampleswere diluted to ensure that all activity measurements fell within thelinear part of the dose-response curve for the assay.

EXAMPLE 2C pH-stability Assay

Suc-AAPF-pNA (Sigma® S-7388) was used for obtaining pH-stabilityprofiles.

Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mMCABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton® X-100 adjusted to pH-values2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or 11.0 withHCl or NaOH.

Each protease sample (in 1 mM succinic acid, 2 mM CaCl₂, 100 mM NaCl, pH6.0 and with an A₂₈₀ absorption>10) was diluted in the assay buffer ateach pH value tested to A₂₈₀=1.0. The diluted protease samples wereincubated for 2 hours at 37° C. After incubation, protease samples werediluted in 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS,1 mM CaCl₂, 150 mM KCl, 0.01% Triton® X-100, pH 9.0, bringing the pH ofall samples to pH 9.0.

In the following activity measurement, the temperature was 25° C.

300 μl diluted protease sample was mixed with 1.5 ml of the pH 9.0 assaybuffer and the activity reaction was started by adding 1.5 ml pNAsubstrate (50mg dissolved in 1.0 ml DMSO and further diluted 45× with0.01% Triton® X-100) and, after mixing, the increase in A₄₀₅ wasmonitored by a spectrophotometer as a measurement of the (residual)protease activity. The 37° C. incubation was performed at the differentpH-values and the activity measurements were plotted as residualactivities against pH. The residual activities were normalized with theactivity of a parallel incubation (control), where the protease wasdiluted to A₂₈₀=1.0 in the assay buffer at pH 9.0 and incubated for 2hours at 5° C. before activity measurement as the other incubations. Theprotease samples were diluted prior to the activity measurement in orderto ensure that all activity measurements fell within the linear part ofthe dose-response curve for the assay.

EXAMPLE 2D Temperature-activity Assay

Protazyme AK tablets were used for obtaining temperature profiles.Protazyme AK tablets are azurine dyed crosslinked casein prepared astablets by Megazyme.

Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mMCABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton® X-100 adjusted to pH 9.0with NaOH.

A Protazyme AK tablet was suspended in 2.0 ml 0.01% Triton X-100 bygentle stirring. 500,1 of this suspension and 500 μl assay buffer weremixed in an Eppendorf tube and placed on ice. 20 μl protease sample(diluted in 0.01% Triton X-100) was added. The assay was initiated bytransferring the Eppendorf tube to an Eppendorf thermomixer, which wasset to the assay temperature. The tube was incubated for 15 minutes onthe Eppendorf thermomixer at its highest shaking rate. By transferringthe tube back to the ice bath, the assay incubation was stopped. Thetube was centrifuged in an ice-cold centrifuge for a few minutes and theA₆₅₀ of the supernatant was read by a spectrophotometer. A buffer blindwas included in the assay (instead of enzyme).A₆₅₀(protease)—A₆₅₀(blind) was a measurement of protease activity. Theassay was performed at different temperatures and the activitymeasurements were plotted as relative activities against incubationtemperature. The relative activities were normalized with the highestactivity (temperature optimum). The protease samples were diluted toensure that all activity measurements fell within the near linear partof the dose-response curve for the assay.

An overview of the activity optima (pH- and temperature activity) isseen in Table 1. pH-stability, pH-activity and temperature-activityprofiles are seen in FIGS. 1-3, and a detailed comparison of thepH-stability data for the proteases at acidic pH-values is seen in Table2.

TABLE 1 pH- and temperature optima of various proteasesTemperature-optimum pH-optimum at pH 9.0 Protease (pNA-substrate)(Protazyme AK) From Bacillus sp. 9 60° C. NCIMB 40484 Sub. Novo¹ 10 70°C. Sub. Novo (Y217L)² 9 70° C. SAVINASE ™³ 9 70° C.

TABLE 2 pH-stability of various proteases, between pH 2.0 and 5.0 pH pHpH pH pH pH pH Protease 2.0 2.5 3.0 3.5 4.0 4.5 5.0 From Bacillus 0.0010.001 0.428 0.940 0.991 0.989 0.991 sp. NCIMB 40484 Sub. Novo 0.0070.003 0.000 0.000 0.024 0.784 0.942 Sub. Novo (Y217L) 0.000 0.000 0.0020.003 0.350 0.951 0.996 Savinase ® 0.001 0.001 0.001 0.003 0.338 0.9290.992

EXAMPLE 2E Absorption Purity of Purified Protease Samples

Determination of A₂₈₀/A₂₆₀ Ratio

The A₂₈₀/A₂₆₀ ratio of purified protease samples is determined asfollows.

A₂₆₀ means the absorption of a protease sample at 260 nm in a 1 cm pathlength cuvette relative to a buffer blank. A₂₈₀ means the absorption ofthe same protease sample at 280 nm in a 1 cm path length cuvetterelative to a buffer blank.

Samples of the purified proteases from Examples 2 and 6 were diluted inbuffer until the A₂₈₀ reading of the spectrophotometer was within thelinear part of its response curve. The A₂₈₀/A₂₆₀ ratio was determinedfrom the readings.

The following results were obtained:

Protease/subtilisin from A₂₈₀/A₂₆₀ Sub. Novo 2.11 Sub. Novo (Y217L) 2.12SAVINASE ™ 2.12 Bacillus sp., NCIMB 40484 2.19 Bacillus alcalophilus,NCIMB 10438 1.92 Fusarium oxysporum, IFO 4471 1.89 Paecilomyceslilacinus, CBS 102449 1.92 Acremonium chrysogenum, ATCC 48272 2.04Acremonium kiliense, ATCC 20338 1.71

EXAMPLE 3 Ability of the Protease Derived from Bacillus sp, NCIMB 40484to Degrade Insoluble Parts of Soy Bean Meal (SBM)

The protease from Bacillus sp. NCIMB 40484 was tested for its ability tomake the insoluble/indigestible parts of SBM accessible to digestiveenzymes and/or added exogeneous enzymes.

Its performance was compared to two aspartate proteases, Protease I andProtease II, prepared as described in WO 95/02044. This document alsodiscloses their use in feed. Protease I is an Aspergillopepsin II typeof protease, and Protease II an Aspergillopepsin I type of protease(both aspartate proteases, ie non-subtilisin proteases) from Aspergillusaculeatus (reference being made to Handbook of Proteolytic Enzymesreferred to above).

The test substrate, the so-called soy remnant, was produced in a processwhich mimics the digestive tract of mono-gastric animals, including apepsin treatment at pH 2, and a pancreatin treatment at pH 7.

In the pancreatin treatment step a range of commercial enzymes was addedin high dosages in order to degrade the SBM components that areaccessible to existing commercial enzymes.

The following enzymes, all commercially available from Novozymes A/S,Denmark, were added: ALCALASE™ 2.4L, NEUTRASE™ 0.5L, FLAVOURZYME™ 1000L,ENERGEX™ L, BIOFEED™ Plus L, PHYTASE NOVO™ L. The SBM used was astandard 48% protein SBM for feed, which had been pelletized.

After the treatment only 5% of the total protein was left in theresulting soy remnant.

FITC Labelling Protocol

The remnant was subsequently labelled with FITC (Molecular Probes,F-143) as follows: Soy remnant (25 g wet, ˜5 g dry) was suspended in 100ml 0.1 M carbonate buffer, pH 9 and stirred 1 hour at 40° C. Thesuspension was cooled to room temperature and treated with fluorescein5-isothiocyanate (FITC) over night in the dark. Non-coupled probe wasremoved by ultrafiltration (10.000 Mw cut-off).

FITC-assay

The FITC-labelled soy remnant was used for testing the ability of theproteases to degrade the soy remnant using the following assay: 0.4 mlprotease sample (with A₂₈₀ =0.1) was mixed with 0.4 ml FITC-soy remnant(suspension of 10 mg/ml in 0.2M sodium-phosphate buffer pH 6.5) at 37°C., and the relative fluorescence units (RFU 485/535 nm;excitation/monitoring wave length) measured after 0 hours, and after 22hours incubation. Before determination of the RFU, samples werecentrifuged for 1 min at 20.000×G and 250 micro-liter supernatant wastransferred to a black micro-titer tray. Measurements were performedusing a VICTOR 1420 Multilabel counter (In vitro, Denmark). RFU isgenerally described by Iain D. Johnson in: Introduction to FluorescenceTechniques, Handbook of Fluorescent Probes and Research Chemicals,Molecular Probes, Richard P. Haugland, 6^(th) edition, 1996 (ISBN0-9652240-0-7).

A blind sample was prepared by adding 0.4 ml buffer instead of enzymesample.RFU_(sample)=ΔRFU_(sample)−ΔRFU_(blind), where ΔRFU=RFU(22 hours)−RFU(0hours)

The resulting FITC values (RFU_(sample) values) are shown in Table 3below. The FITC values are generally with an error margin of +/−20.000.Contrary to Protease I and Protease II, the protease derived fromBacillus sp. NCIMB 40484 degraded the soy remnant to a significantextent.

TABLE 3 Ability of proteases to degrade soy remnant Protease FITC(+/−20000) Bacillus sp. NCIMB 40484 61900 Protease I −9200 Protease II−1200

EXAMPLE 4 In vitro Testing of the Protease Derived from Bacillus sp.NCIMB 40484

The protease derived from Bacillus sp. NCIMB 40484 was tested togetherwith other subtilisin proteases such as SubNovo, SubNovo (Y217L),SAVINASE™ and ALCALASE™ for its ability to solubilise maize-SBM(maize-Soy Bean Meal) proteins in an automated in vitro digestion system(simulating digestion in monogastric animals). For the blank treatments,maize-SBM was incubated in the absence of exogenous subtilisin-likeproteases.

The in vitro system consisted of 30 flasks in which maize-SBM substratewas initially incubated with HCl/pepsin—simulating gastric digestion—andsubsequently with pancreatin—simulating intestinal digestion. At the endof the gastric incubation period samples of in vitro digesta wereremoved and analysed for solubilised protein.

Substrates

10 g maize-SBM diet with a maize-SBM ratio of 6:4 (w/w) was used. Theprotein content was 43% (w/w) in SBM and 8.2% (w/w) in maize meal. Thetotal amount of protein in 10 g maize-SBM diet was 2.21 g.

Digestive Enzymes

Pepsin (Sigma P-7000; 539 U/mg, solid), pancreatin (Sigma P-7545;8xU.S.P. (US Pharmacopeia)).

Outline of In vitro Digestion Procedure

Simulated Time digestion Components added to flask pH Temp. course phase10 g maize-SBM diet (6:4), 3.0 40° C. t = 0  Gastric HCl/pepsin (3000U/g min diet), protease (0.1 mg protease enzyme protein/g diet) NaOH 6.840° C. t = 60 Intestinal min NaHCO₃/pancreatin (8 mg/g 6.8 40° C. t = 90diet) min Stop incubation, remove 7.0  0° C.  t = 330 aliquot minEnzyme Protein Determinations

The amount of protease enzyme protein is calculated on the basis of theA₂₈₀ values and the amino acid sequences (amino acid compositions) usingthe principles outlined in S. C. Gill & P. H. von Hippel, AnalyticalBiochemistry 182, 319-326, (1989).

Experimental Procedure for In vitro Model

1. 10 g of substrate is weighed into a 100 ml flask.

2. At time 0 min, 46 ml HCl (0.1 M) containing pepsin (3000 U/g diet)and 1 ml of protease (0.1 mg enzyme protein/g diet) are added to theflask while mixing. The flask is incubated at 40° C.

3. At time 30 min, pH is measured.

4. At time 45 min, 16 ml of H₂O is added.

5. At time 60 min, 7 ml of NaOH (0.39 M) is added.

6. At time 90 min, 5 ml of NaHCO₃ (1M) containing pancreatin (8.0 mg/gdiet) is added.

7. At time 120 min, pH is measured.

8. At time 300 min, pH is measured.

9. At time 330 min, samples of 30 ml are removed and placed on icebefore centrifugation (10000×g, 10 min, 4° C.). Supernatants are removedand stored at −20° C.

Estimation of Solubilised Protein by Gelfiltration HPLC

The content of solubilised protein in supernatants from in-vitrodigested samples was estimated by quantifying crude protein (CP) usinggelfiltration HPLC. Supernatants were thawed, filtered through 0.45 μmpolycarbonate filters (Sar-torius) and diluted (1:50, v/v) with H₂O.Diluted samples were chromatographed by HPLC using a Superdex Peptide PE(7.5×300 mm) gelfiltration column (Global). The eluent used forisocratic elution was 50 mM sodium phosphate buffer (pH 7.0) containing150 mM NaCl. The total volume of eluent per run was 26 ml and the flowrate was 0.4 ml/min. Elution profiles were recorded at 214 nm and thetotal area under the profiles was determined by integration. To estimateprotein content from integrated areas, a calibration curve (R²=0.9993)was made from a dilution series of an in vitro digested referencemaize-SBM sample with known total protein content. The proteindetermination in this reference sample was carried out by the Kjeldahlmethod (determination of % nitrogen; A.O.A.C. (1984) Official Methods ofAnalysis 14^(th) ed., Washington DC).

Results

The results, i.e. the effect of the various proteases on proteinsolubility in vitro, are shown in Table 4 below.

The calculation of relative amounts of solubilised protein is based onthe total amount of protein in 10 g maize-SBM diet (2.21 g protein)dissolved in a total volume of 75 ml during the in vitro digestionreaction. Assuming complete protein solubilisation (100%), the proteincontent in supernatants would be 2.95% weight per volume.

The results were analysed by one-way analysis of variance: P=0.0001).SD=Standard Deviation; n=the number of replicas per treatment (n=5).

The protease derived from Bacillus sp. NCIMB 40484 has a significantlybetter effect on protein solubilisation as compared to the otherproteases.

TABLE 4 Soluble CP Enzyme (% of total) SD Protease from Bacillus sp.NCIMB 78.8^(A) 0.48 40484 Sub. Novo 76.7^(B) 0.37 ALCALASE ™ 73.9^(C)1.04 Sub. Novo (Y217L) 75.8^(B) 0.91 SAVINASE ™ 75.8^(B) 0.85 Blank76.6^(B) 0.88 ^(A, B, C)Values not sharing a common index letter differsignificantly (P < 0.05)

EXAMPLE 5 Degradation of the Lectin SPA and the Soybean Bowman-Birk andKunitz Inhibitors

The ability of the protease from Bacillus sp. NCIMB 40484 to hydrolysesoybean agglutinin (SBA) and the soy Bowman-Birk and Kunitz trypsininhibitors was tested.

Pure SBA (Fluka 61763), Bowman-Birk Inhibitor (Sigma T-9777) or KunitzInhibitor (Trypsin Inhibitor from soybean, Boehringer Mannheim 109886)was incubated with the protease for 2 hours, 37° C., at pH 6.5(protease: anti-nutritional factor=1:10, based on A₂₈₀). Incubationbuffer: 50 mM dimethyl glutaric acid, 150 mM NaCl, 1 mM CaCl₂, 0.01%Triton X-100, pH 6.5.

The ability of the protease to degrade SBA and the protease inhibitorswas estimated from the disappearance of the native SBA or trypsininhibitor bands and appearance of low molecular weight degradationproducts on SDS-PAGE gels. Gels were stained with Coomassie blue andband intensity determined by scanning.

The results, as % of anti-nutritional factor degraded, are shown inTable 5 below.

It is contemplated that the ability to degrade the anti-nutritionalfactors in soy can also be estimated by applying the Western techniquewith antibodies against SBA, Bowman-Birk Inhibitor or Kunitz Inhibitorafter incubation of soybean meal with the candidate proteases (seeWO98/56260).

TABLE 5 Protease Bowman-Birk Kunitz derived from SBA Inhibitor InhibitorBacillus sp. 21 41 100 NCIMB 40484

EXAMPLE 6 Preparation of Further Acid-stable Subtilisins Preparation ofthe Bacillus alcalophilus protease

Bacillus alcalophilus NCIMB 10438 was inoculated from a freeze driedculture into shake flasks each containing 100 ml BPX medium with thefollowing composition: potato starch 100 g/l, barley flour 50 g/l, BAN800 MG (obtainable from Novozymes A/S) 0.05 g/l, sodium caseinate 10g/l, soy meal 20 g/l, di-sodiumphosphate 9 g/l, Pluronic PE 6100 0.1ml/l in tap water. The pH was adjusted to 9.7 with 10 ml 1M sodiumsesquicarbonate in each shake flask before inoculation. The strain wasfermented for 4 days at 30 degree C. at 300 rpm. From this culture newshake flasks containing 100 ml BPX medium were inoculated and fermentedfor 3 days.

Purification

The culture broth was centrifuged at 10000×g for 30 minutes in 1 literbeakers. The supernatants were combined and further clarified by afiltration though a Seitz K-250 depth filter plate. The clear filtratewas concentrated by ultrafiltration on a 3 kDa cut-off polyether sulfonecassette (Filtron). The concentrated enzyme was transferred to 50 mMH₃BO₃, 5 mM 3,3′-dimethyl glutaric acid, 1 mM CaCl₂, pH 7 (Buffer A) ona G25 Sephadex column (Amersham Pharmacia Biotech), and applied to aBacitracin agarose column (Upfront Chromatography A/S) equilibrated inBuffer A. After washing the Bacitracin column with Buffer A to removeunbound protein, the protease was eluted from the column using Buffer Asupplemented with 25% 2-propanol and 1M sodium chloride. The fractionsfrom the Bacitracin column with protease activity were pooled andtransferred to 20 mM CH₃COOH/NaOH, 1 mM CaCl₂, pH 5 (Buffer B) by G25Sephadex chromatography. The buffer exchanged protease pool was appliedto a SOURCE 30S column (Amersham Pharmacia Biotech) equilibrated inBuffer B. After washing the SOURCE 30S column with Buffer B, theprotease was eluted with an increasing linear NaCl gradient (0 to 0.5M)in Buffer B. Fractions from the column were tested for protease activityand protease containing fractions were analysed by SDS-PAGE. Purefractions were pooled and used for further characterisation.

Preparation of Other Acid-stable Subtilisins

The proteases of Fusarium oxysporum IFO 4471, Bacillus alcalophilusNCIMB 10438, Paecilomyces lilacinus CBS 102449, Acremonium chrysogenumATCC 48272, and Acremonium kiliense ATCC 20388 were prepared usingconventional methods, as generally described above for the protease ofBacillus alcalophilus, NCIMB 10438.

Sequences

The following partial amino acid sequences were determined:

SEQ ID NO: 1

-   -   N-terminal of the protease derived from Acremonium chrysogenum        ATCC 48272: ALVTQNGAPWGLGTISHRQPGSTSYIY;

SEQ ID NO: 2

-   -   N-terminal of the protease derived from Bacillus alcalophilus        NCIMB 10438: NQVTPWGITRVQAPTAW;

SEQ ID NO: 3

-   -   N-terminal of the protease derived from Paecilomyces lilacinus        CBS 102449: AYTQQPGAPWGLGRISH;

SEQ ID NO: 4

-   -   N-terminal of the protease derived from Fusarium oxysporum IFO        4471: ALTTQSGATWGLGTVSHRSRGS.

The amino acid sequence of the protease derived from Bacillus sp. NCIMB40484, SEQ ID NO: 5 herein, had been previously determined (see U.S.Pat. No. 5,650,326, SEQ ID NOs: 4, 6 and 8).

A search in public protein databases for related sequences revealed thefollowing:

SEQ ID NO: 6

-   -   Geneseqp/r65936 (referring to the protease of Paecilomyces        lilacinus CBS 143.75 of EP 623672)—related to SEQ ID NO: 3;

SEQ ID NO: 7

-   -   Geneseqp/r74334 (referring to the protease of Bacillus sp.        THS-1001 of JP-07095882)—related to SEQ ID NO: 2.

The strains of Paecilomyces lilacinus and Aspergillus sp. have beendeposited according to the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure at the Centraalbureau voor Schimmelcultures (CBS), P.O Box273, 3740 AG Baarn, The Netherlands, as follows.

Deposit date Jan. 17, 2000 CBS No. Aspergillus sp. 102448 Deposit dateJan. 17, 2000 CBS No. Paecilomyces lilacinus 102449

EXAMPLE 7 Characterisation and Comparative Study of Further SubtilisinProteases

The proteases prepared from Bacillus alcalophilus NCIMB 10438, Fusariumoxysporum IFO 4471, Paecilomyces lilacinus CBS 102449, Acremoniumchrysogenum ATCC 48272, Acremonium kiliense ATCC 20338 are allsubtilisins.

The purity of the protease samples was determined as described inexample 2.

The following results were obtained:

SDS-PAGE Protease Purity (Area %) Bacillus alcalophilus NCIMB 10438100.0  Fusarium oxysporum IFO 4471 n.d. Paecilomyces lilacinus CBS102449 98.3 Acremonium chrysogenum ATCC 48272 98.6 Acremonium kilienseATCC 20338 n.d. n.d. = not determinedAssays

The pH-activity, pH-stability and temperature-activity assays aredescribed in Example 2 (the pNA substrate Suc-AAPF-pNA (Sigma S-7388)was used for all of the proteases for pH-activity and -stabilityprofiles, whereas Protazyme AK tablets were used for the temperatureprofiles).

An overview of the activity optima (pH- and temperature activity) isseen in Table 6. pH-stability, pH-activity and temperature-activityprofiles are seen in FIGS. 4-6, and a detailed comparison of thepH-stability data for the proteases at acidic pH-values is seen in Table7.

TABLE 6 pH- and temperature optima of various proteases pH- Temperature-Protease optimum optimum (° C.) Bacillus alcalophilus NCIMB 10438 9 70Fusarium oxysporum IFO 4471 11 60 Paecilomyces lilacinus CBS 102449 8 60Acremonium chrysogenum ATCC 48272 9 70 Acremonium kiliense ATCC 20338 1170

TABLE 7 pH-stability of various proteases, between pH 2.0 and 5.0 pHProtease 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Bacillus 0.007 0.005 0.175 0.8440.965 1.017 1.038 alcalophilus NCIMB 10438 Fusarium 0.000 0.000 0.0030.649 0.929 1.030 1.056 oxysporum IFO 4471 Paecilomyces 0.002 0.0030.005 0.450 0.897 1.000 0.947 lilacinus CBS 102449 Acremonium 0.0020.001 0.001 0.809 0.894 0.972 1.005 chrysogenum ATCC 48272 Acremonium0.008 0.003 0.023 0.412 0.843 0.955 1.009 kiliense ATCC 20338

EXAMPLE 8 Inhibition of Proteases with Streptomyces Subtilisin Inhibitor(SSI)

pNA substrate: Suc-AAPF-pNA (Sigma® S-7388) was used for measuringresidual activity after inhibition.

Assay buffer: 100 mM succinic acid (Merck 1.00682), 100 mM HEPES (SigmaH-3375), 100 mM CHES (Sigma C-2885), 100 mM CABS (Sigma C-5580), 1 mMCaCl₂, 150 mM KCl, 0.01% Tritone X-100, pH 9.0.

Assay temperature: 25° C.

SSI was purified from a Streptomyces albogriseolus FERM P-1205 (S-3253)fermentation supernatant by chromatography. The used SSI preparation hada purity above 95%—the purity was determined by the procedure describedin Example 2A. Alternatively, SSI can be obtained from Wako in Japan,catalog no. 303-05201, manufactured by Daiwa Kasei K.K.

Before the below inhibition assay, SSI was diluted in 0.01% Triton X-100to A₂₈₀ concentration=0.010.

Protease: The used protease had a purity above 95%—the purity wasdetermined by the procedure described in Example 2A. Before the belowinhibition assay, the protease was diluted in 0.01% Triton X-100 to A₂₈₀concentration=0.010.

The inhibition of the proteases by the Streptomyces Subtilisin Inhibitor(SSI) was determined by the following procedure:

A 300 μl protease sample (A₂₈₀ concentration=0.010) was mixed with 300μl SSI (A₂₈₀ concentration=0.010) and 1.5 ml Assay buffer. After 15minutes incubation at room temperature, the residual activity wasmeasured by adding 1.5 ml pNA substrate (50 mg dissolved in 1.0 ml DMSOand further diluted 45× with 0.01% Triton® X-100) and, after mixing, theincrease in A₄₀₅ was monitored by a spectrophotometer. As a control (noSSI), 300 μl 0.01% Triton X-100 was used instead of SSI.

The residual activity was normalized with the control activity (no SSI),i.e. no inhibition by SSI will give 100% residual activity and fullinhibition by SSI will give 0% residual activity.

The following results were obtained:

Residual activity Protease, subtilisin from (%) Bacillus sp., NCIMB40484 4.3 Bacillus amyloliquefaciens 0.1 Bacillus amyloliquefaciens(Y217L) 0.0 Bacillus clausii, (Savinase ®) 0.0 Bacillus alcalophilus,NCIMB 10438 0.0 Fusarium oxysporum IFO 4471 0.1 Paecilomyces lilacinus,CBS 102449 0.1 Acremonium chrysogenum, ATCC 48272 0.1 Acremoniumkiliense, ATCC 20338 n.d.* *not determined

EXAMPLE 9 Ability of Further Acid-stable Subtilisins to DegradeInsoluble Parts of Soy Bean Meal (SBM)

The further acid-stable subtilisins prepared as described in Example 6were tested as described in Example 3 for their ability to make theinsoluble/indigestible parts of SBM accessible to digestive enzymesand/or added exogeneous enzymes.

The results obtained are shown in Table 8 below. For comparison, theresults obtained in Example 3 for proteases I and II are included alsoin Table 8.

TABLE 8 Ability of further proteases to degrade soy remnant Protease,subtilisin from FITC/(+/−20000) Bacillus alcalophilus  81300 NCIMB 10438Fusarium oxysporum IFO 4471 102200 Paecilomyces lilacinus  98700 CBS102449 Acremonium chrysogenum  89600 ATCC 48272 Acremonium kiliense 94600 ATCC 20338 Protease I  −9200 Protease II  −1200

EXAMPLE 10 Effects of the Acid-stable Subtilisin Derived from Bacillussp. NCIMB 40484 on the Growth Performance of Broiler Chickens

The trial was carried out at the Roche Research Center for AnimalNutrition (CRNA, F-68305 Village-Neuf, France) in accordance with theofficial French instructions for experiments with live animals. Day-oldbroiler chickens (‘Ross PM3’), separated by sex, were supplied by acommercial hatchery.

The chickens were housed in wire-floored battery cages, which were keptin an environmentally controlled room. Feed and tap water was providedad libitum.

On day 8, the chickens were divided by weight into groups of 6 birds,which were allocated to either the control treatment, receiving theexperimental diet without enzymes, or to the enzyme treatment, receivingthe experimental diet supplemented with 100 mg enzyme protein of theBacillus sp. NCIMB 40484 protease per kg feed.

Each treatment was replicated with 12 groups, 6 groups of each sex. Thegroups were weighed on days 8 and 29. The feed consumption of theintermediate period was determined and body weight gain and feedconversion ratio were calculated.

The experimental diet based on maize starch and soybean meal (44% crudeprotein) as main ingredients (Table 9) was produced in the CRNA. Thefeed was pelleted (die configuration: 3×20 mm) at about 70° C. Anappropriate amount of the Bacillus sp. NCIMB 40484 protease was dilutedin a fixed quantity of water and sprayed onto the pelleted feed. For thecontrol treatment, adequate amounts of water were used to handle thetreatments in the same way.

For the statistical evaluation, a two factorial analysis of variance(factors: treatment and sex) was carried out, using the GLM procedure ofthe SAS package (SAS Institute Inc., 1985). Where significant treatmentseffects (p<0.05) were indicated, the differences between treatment meanswere analyzed with the Duncan test. Due to technical reasons, one cageof the enzyme treatment was excluded from the statistical evaluation.

In Table 2 the results of the growth performance of the broiler chickensfrom day 8 to day 29 are listed. There were no interactions betweentreatment and sex, therefore the pooled results of both sexes arepresented. The supplementation of the experimental diet with Bacillussp. NCIMB 40484 protease improved weight gain numerically by 6.6%. Theaddition of the protease increased the feed intake slightly by 3.1%.Bacillus sp. NCIMB 40484 protease improved the feed conversion of thebroiler chickens significantly by 3.4%.

Taking into consideration that maize starch is a highly digestibleingredient, it can be assumed that the observed effects were mainly dueto the action of the enzymes on the soybean meal. Therefore, the resultsindicated that the nutritive value of the soybean meal was improved bythe Bacillus sp. NCIMB 40484 protease.

In conclusion, the study demonstrated that supplementation of broilerfeed containing high amounts of soybean meal with the Bacillus sp. NCIMB40484 protease at 100 mg enzyme protein/kg feed resulted in a numericalincrease of weight gain and a significant improvement of feedconversion.

References

-   EEC (1986): Directive de la Commission du 9 avril 1986 fixant la    méthode de calcul de la valeur énérgetique des aliments composés    destinés à la volaille. Journal Officiel des Communautés    Européennes, L130, 53-54-   SAS Institute Inc. (1985): SAS® User's Guide, Version 5 Edition.    Cary N.C.

TABLE 9 Composition of the experimental diet Ingredients (%): Maizestarch 45.80 Soybean meal 44 ¹ 44.40 Tallow 3.20 Soybean oil 1.00DL-Methionine 0.18 MCP 0.76 Salt 0.05 Binder 1.00 Vitamin and mineralpremix 3.55 Avatec ® 15% CC ² 0.06 Analyzed content: Crude protein (%)19.3 ME, N-corrected (MJ/kg) ³ 12.2 Crude fat (%) 5.3 ¹ analyzedcontent: 90.6% dry matter, 45.3% crude protein, 2.0% crude fat, 4.9%crude fibre ² corresponded to 90 mg lasalocid-Na/kg feed asanticoccidial ³ calculated on the basis of analyzed nutrients content(EC-equation; EEC, 1986)Supplier of Feed Ingredients

-   Maize starch: Roquettes Frères, F-62136 Lestrem, France-   Soybean meal 44: Rekasan GmbH, D-07338 Kaulsdorf, Germany-   Tallow: Fondoirs Gachot SA, F-67100 Strasbourg, France-   Soybean oil: Ewoco Sarl, F-68970 Guemar, France-   DL-Methionine: Produit Roche SA, F-92521 Neuilly-sur-Seine, France-   MCP: Brenntag Lorraine, F-54200 Toul, France-   Salt: Minoterie Moderne, F-68560 Hirsingue, France-   Binder: Minoterie Moderne, F-68560 Hirsingue, France-   Premix (AM vol chair NS 4231): Agrobase, F-01007 Bourg-en-Bresse,    France-   Avatec: Produit Roche SA, F-92521 Neuilly-sur-Seine, France

TABLE 10 Performance of broiler chickens from days 8 to 29 Pooledresults of both sexes; mean ± st. dev. Product Control Bacillus sp.NCIMB 40484 pro- tease Dose per kg   0  100 mg enzyme feed protein Cages× birds 12 × 6 11 × 6 ¹ Weight gain 1155 ^(A) ± 1231 ^(A) ± (g/bird)  94 98 (%)  100.0  106.6 Feed intake 1941 ^(A) ± 2002 ^(A) ± (g/bird)  108 145 (%)  100.0  103.1 Feed conversion   1.684 ^(A) ±   1.627 ^(B) ± (gfeed/g gain)   0.069   0.031 (%)  100.0  96.6 Means within a row, notsharing a common superscript are significantly different (p < 0.05) ¹Due to technical reasons, one cage was excluded from the statisticalevaluation

EXAMPLE 11 Premix and Diets for Turkey and Salmonids Supplemented withAcid-stable Subtilisin Protease

A premix of the following composition is prepared (content per kilo):

5000000 IE Vitamin A 1000000 IE Vitamin D3  13333 mg Vitamin E   1000 mgVitamin K3   750 mg Vitamin B1   2500 mg Vitamin B2   1500 mg Vitamin B6  7666 mg Vitamin B12  12333 mg Niacin  33333 mg Biotin   300 mg FolicAcid   3000 mg Ca-D-Panthothenate   1666 mg Cu  16666 mg Fe  16666 mg Zn 23333 mg Mn   133 mg Co    66 mg I    66 mg Se    5.8% Calcium

To this premix is added Bacillus sp. NCIMB 40484 protease prepared asdescribed in Example 2 in an amount corresponding to 10 g proteaseenzyme protein/kg.

Pelleted turkey starter and grower diets with a composition as shown inthe below table (on the basis of Leeson and Summers, 1997 butrecalculated without meat meal by using the AGROSOFT®, optimisationprogram) and with 100 mg protease enzyme protein per kg are prepared asfollows:

Milled maize, Soybean meal, Fish-meal and Vegetable fat are mixed in acascade mixer. Limestone, calcium phosphate and salt are added, togetherwith the above premix in an amount of 10 g/kg diet, followed by mixing.The resulting mixture is pelleted (steam conditioning followed by thepelleting step).

Starter Grower, Ingredient diet, g/kg g/kg Finisher Maize 454.4 612.5781.0 Soybean meal 391 279 61.7 Fish meal 70 29.9 70 Vegetable fat 21 2146 Limestone 19 16.9 9 Calcium phosphate 30 25.9 16.8 Salt (NaCl) 2 2 2Vitamin and mineral 10 10 10 premix Lysine 1.3 1.49 Methionine 1.3 1.33.6 Calculated nutrients Crude protein g/kg 279 213 152 Metabolisableenergy 12.3 12.7 14.1 MJ/kg Calcium, g/kg 15.8 12.7 9 AvailablePhosphorus, 8.2 6.4 4.6 g/kg Lysine, g/kg 17.6 12.8 7.5 Methionine, g/kg6.1 4.9 6.9

Two diets for Salmonids are also prepared, as generally outlined above.The actual compositions are indicated in the Table below (compiled fromRefstie et al (1998), Aquaculture, vol. 162, p.301-302). The estimatednutrient content is recalculated by using the Agrosoft™ feedoptimisation program.

The protease derived from Bacillus sp. NCIMB 40484, prepared asdescribed in Example 2, is added to the diets in an amount correspondingto 100 mg protease enzyme protein per kg.

Conventional diet Alternative diet Ingredient with fish meal withsoybean meal Wheat 245.3 75.2 Fish meal 505.0 310.0 Soybean meal — 339.0Fish oil 185.0 200.0 DL-Methionine 13.9 23.0 Mono-Calcium phos- — 2.0phate Vitamin and Mineral 50.8 50.8 premix + pellet binder and astaxan-thin Calculated nutrients (fresh weight basis) Crude protein g/kg 401415 Crude fat g/kg 232 247 Metabolisable energy 16.9 16.5 MJ/kg Calcium,g/kg 13.9 9.8 Phosphorus, g/kg 10.8 9.0 Lysine, g/kg 27.7 26.7Methionine, g/kg 24.4 31.6

EXAMPLE 12 Determination of Purity of Protease-containing EnzymeProducts

The purity of protease-containing enzyme products, e.g. proteasepreparations such as commercial multi-component enzyme products, can bedetermined by a method based on the fractionation of theprotease-containing enzyme product on a size-exclusion column.Size-exclusion chromatography, also known as gel filtrationchromatography, is based on a porous gel matrix (packed in a column)with a distribution of pore sizes comparable in size to the proteinmolecules to be separated. Relatively small protein molecules candiffuse into the gel from the surrounding solution, whereas largermolecules will be prevented by their size from diffusing into the gel tothe same degree. As a result, protein molecules are separated accordingto their size with larger molecules eluting from the column beforesmaller ones.

Protein Concentration Assay

The protein concentration in protease-containing enzyme products isdetermined with a BCA protein assay kit from PIERCE (identical to PIERCEcat. No. 23225). The sodium salt of Bicinchoninic acid (BCA) is astable, water-soluble compound capable of forming an intense purplecomplex with cuprous ions (Cu¹⁺) in an alkaline environment. The BCAreagent forms the basis of the BCA protein assay kit capable ofmonitoring cuprous ions produced in the reaction of protein withalkaline Cu²⁺ (Biuret reaction). The colour produced from this reactionis stable and increases in a proportional fashion with increasingprotein concentrations (Smith, P. K., et al. (1985), AnalyticalBiochemistry, vol. 150, pp. 76-85). The BCA working solution is made bymixing 50 parts of reagent A with 1 part reagent B (Reagent A is PIERCEcat. No. 23223, contains BCA and tartrate in an alkaline carbonatebuffer; reagent B is PIERCE cat. No. 23224, contains 4% CuSO₄*5H₂O). 300μl sample is mixed with 3.0 ml BCA working solution. After 30 minutes at37° C., the sample is cooled to room temperature and A₄₉₀ is read as ameasure of the protein concentration in the sample. Dilutions of Bovineserum albumin (PIERCE cat. No. 23209) are included in the assay as astandard.

Sample Pre-treatment

If the protease-containing enzyme product is a solid, the product isfirst dissolved/suspended in 20 volumes of 100 mM H₃BO₃, 10 mM3,3′-dimethylglutaric acid, 2 mM CaCl₂, pH 6 (Buffer A) for at least 15minutes at 5° C., and if the enzyme at this stage is a suspension, thesuspension is filtered through a 0.45μ filter to give a clear solution.The solution is from this point treated as a liquid protease-containingenzyme product.

If the protease-containing enzyme product is a liquid, the product isfirst dialysed in a 6-8000 Da cut-off SpectraPor dialysis tube (cat.no.132 670 from Spectrum Medical Industries) against 100 volumes of BufferA+150 mM NaCl (Buffer B) for at least 5 hours at 5° C., to removeformulation chemicals that could give liquid protease-containing enzymeproducts a high viscosity, which is detrimental to the size-exclusionchromatography.

The dialysed protease-containing enzyme product is filtered through a0.45μ filter if a precipitate was formed during the dialysis. Theprotein concentration in the dialysed enzyme product is determined withthe above described protein concentration assay and the enzyme productis diluted with Buffer B, to give a sample ready for size-exclusionchromatography with a protein concentration of 5 mg/ml. If the enzymeproduct has a lower than 5 mg/ml protein concentration after dialysis,it is used as is.

Size-exclusion Chromatography

A 300 ml HiLoad26/60 Superdex75pg column (Amersham Pharmacia Biotech) isequilibrated in Buffer B (Flow: 1 ml/min). 1.0 ml of theprotease-containing enzyme sample is applied to the column and thecolumn is eluted with Buffer B (Flow: 1 ml/min). 2.0 ml fractions arecollected from the outlet of the column, until all of the applied samplehave eluted from the column. The collected fractions are analysed forprotein content (see above Protein concentration assay) and for proteaseactivity by appropriate assays. An example of an appropriate assay isthe Suc-AAPF-pNA assay (see Example 2B). Other appropriate assays aree.g. the CPU assay (se Example 1), and the Protazyme AK assay (seeExample 2D). The conditions, e.g. pH, for the protease activity assaysare adjusted to measure as many proteases in the fractionated sample aspossible. The conditions of the assays referred to above are examples ofsuitable conditions. Other suitable conditions are mentioned above inthe section dealing with measurement of protease activity. A proteinpeak with activity in one or more of the protease assays is defined as aprotease peak. The purity of a protease peak is calculated as theprotein amount in the peak divided with the total protein amount in allidentified protease peaks.

The purity of a protease-containing enzyme product is calculated as theamount of protein in the acid-stable protease peak divided with theprotein amount in all identified protease peaks using the aboveprocedure.

1. An animal feed additive comprising (a) at least one purifiedacid-stable subtilisin and (b) one or more fat soluble vitamins and/orwater soluble vitamins, and (c) one or more trace minerals, wherein theacid stability of the subtilisin means that the activity of the puresubtilisin, in a dilution corresponding to A₂₈₀=1.0, is at least 40% ofthe reference activity of the subtilisin, wherein the activity of thesubtilisin is measured after two hours incubation at a temperature of37° C. in a buffer of 100 mM succinic acid, 100 mM HEPES, 100 mM CHES,100 mM CABS, 1 mM CaCl₂, 150 M KCl, and 0.01% Triton X-100 (pH 3.5); andwherein the reference activity is measured after two hours incubation ata temperature of 5° C. in the same buffer but adjusted to pH 9.0,wherein the activity and reference activity are measured after theseincubations, at 25° C. in Suc-AAPF-pNA (pH 9.0).
 2. The animal feedadditive of claim 1, wherein the activity of the pure subtilisin is atleast 45% of the reference activity of the subtilisin.
 3. The animalfeed additive of claim 2, wherein the activity of the subtilisin is atleast 50% of the reference activity.
 4. The animal feed additive ofclaim 3, wherein the activity of the subtilisin is at least 60% of thereference activity.
 5. The animal feed additive of claim 1, wherein thesubtilisin has a pH optimum in the range of 6.0-11.0.
 6. The animal feedadditive of claim 1, wherein the subtilisin is a Bacillus sp., NCIMB40484 subtilisin.
 7. The animal feed additive of claim 1, wherein thesubtilisin is a Bacillus alcalophilus NCIMB 10438 subtilisin.
 8. Theanimal feed additive of claim 1, wherein the subtiuisin is a Fusariumoxysporum IFO 4471 subtilisin.
 9. The animal feed additive of claim 1,wherein the subtilisin is a Paecilomyces lilacinus CBS 102449subtilisin.
 10. The animal feed additive of claim 1, wherein thesubtilisin is an Acremonium chrysogenum ATCC 48272 subtilisin.
 11. Theanimal feed additive of claim 1, wherein the subtilisin is an Acremoniumkiliense ATCC 20338 subtilisin.
 12. The animal feed additive of claim 1,wherein the amount of the purified subtilisin corresponds to 0.01-200 mgsubtilisin protein per kg feed.
 13. An animal feed additive of claim 1,for addition to an animal feed having a crude protein content of 50-800g/kg.
 14. The animal feed additive of claim 1, which further comprisesat least one enzyme selected from the group consisting of phytase,xylanase, galactanase, and beta-glucanase.
 15. A method for theimproving the nutritional value of a vegetable protein or proteinsource, comprising adding an animal feed additive of claim 1 to thevegetable protein or protein source.
 16. The method of claim 15, whereinthe vegetable protein comprises soybean.
 17. The method of claim 15,wherein the subtilisin is a Bacillus sp., NCIMB 40484 subtilisin. 18.The method of claim 15, wherein the subtilisin is a Bacillusalcalophilus, NCIMB 10438 subtilisin.
 19. The method of claim 15,wherein the subtilisin is a Fusarium oxysporum, IFO 4471 subtilisin. 20.The method of claim 15, wherein the subtilisin is a Paecilomyceslilacinus, CBS 102449 subtilisin.
 21. The method of claim 15, whereinthe subtilisin is an Acremonium chrysogenum, ATCC 48272 subtilisin. 22.The method of claim 15, wherein the subtilisin is an Acremoniumkiliense, ATCC 20338 subtilisin.